CANCER STEM CELLS

Connexin 43 Reverses Malignant Phenotypes of Glioma Stem Cells

by Modulating E-Cadherin

SHI-CANG YU,aHUA-LIANG XIAO,

aXUE-FENG JIANG,

aQING-LIANG WANG,

aYAN LI,

bXIAO-JUN YANG,

a

YI-FANG PING,a JIANG JIE DUAN,a JIAN-YONG JIANG,a XIAN-ZONG YE,a SEN-LIN XU,a YANG-HONG XIN,a

XIAO-HONG YAO,a JIAN-HONG CHEN,a WEI-HUA CHU,c WEI SUN,d BING WANG,a JI MING WANG,e

XIA ZHANG,aXIU-WU BIAN

a

aInstitute of Pathology and Southwest Cancer Center, bCentral Laboratory, cDepartment of Neurosurgery,

Southwest Hospital, dCentral Laboratory, Third Military Medical University, Chongqing, China; eLaboratory of

Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer

Institute at Frederick, Frederick, Maryland, USA

Key Words. Connexin 43 • Glioma stem cells • E-Cadherin • Gap junctional intercellular communication • Invasiveness • Tumorigenicity

ABSTRACT

Malfunctioned gap junctional intercellular communication

(GJIC) has been thought associated with malignant trans-formation of normal cells. However, the role of GJIC-

related proteins such as connexins in sustaining the malig-nant behavior of cancer stem cells remains unclear. Inthis study, we obtained tumorspheres formed by glioma

stem cells (GSCs) and adherent GSCs and then examinedtheir GJIC. All GSCs showed reduced GJIC, and differ-entiated glioma cells had more gap junction-like struc-

tures than GSCs. GSCs expressed very low level ofconnexins, Cx43 in particular, which are key components

of gap junction. We observed hypermethylation in the

promoter of gap junction protein a1, which encodes Cx43in GSCs. Reconstitution of Cx43 in GSCs inhibited their

capacity of self-renewal, invasiveness, and tumorigenicityvia influencing E-cadherin and its coding protein, whichleads to changes in the expression of Wnt/b-catenin target-

ing genes. Our results suggest that GSCs require the lowexpression of Cx43 for maintaining their malignant phe-notype, and upregulation of Cx43 might be a potential

strategy for treatment of malignant glioma. STEM CELLS

2012;30:108–120

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Biological processes in multicellular organisms are regulatedaccurately by the substantial exchange of feedback informa-tion transferring in complicated networks [1]. Gap junctionalintercellular communication (GJIC) through gap junction,which is composed of a family of more than 20 connexins(Cxs), is the most important format of direct communicationbetween adjacent cells. Gap junction allows direct intercellu-lar exchange of ions (e.g., Ca2þ), second messengers, smallmetabolites, and peptides for normal function of cells [2–6].Signals of contact inhibition, apoptosis, differentiation, andlocalization are transferred from adjacent cells through gapjunction to maintain cellular homeostasis [2, 3].

Uncontrollable proliferation, poor differentiation, and highinvasiveness are biological features of cancers. It has beenreported that dysfunction of GJIC may affect cell prolifera-tion, differentiation, and localization [7]. For instance, mal-

function and disruption of GJIC are correlated with tumori-genesis in pulmonary and hepatic cells [8–10]. Severalcarcinogens are shown to decrease Cxs expression in normalepithelial cells [11], while enhancement of GJIC function hasa profound effect for growth inhibition of cancers such as gli-oma with unknown mechanism [12–19]. Overall results fromthe studies of relationship between Cxs and glioma invasionhave been controversial [20, 21].

Cancer stem cells (CSCs), although as a small fraction ofcancer cell population, have been believed to play crucialroles in tumor malignancy. The isolation and characterizationof CSCs have been assisting our better understanding of tu-mor initiation, progression, and recurrence [22–24]. Accumu-lating evidence from our and other laboratories has showedthat CSCs in malignant glioma, that is, glioma stem cells(GSCs), possess the capacities of indefinite proliferation, dif-ferentiation multipotency, and higher tumorigenicity [25–40].GSCs are more aggressive than the committed glioma cells.Therefore, GSCs might be the resources of invasiveness and

Author contributions: S.-C.Y.: conception and design, performance of experiments, data analysis and interpretation, and manuscriptwriting; H.L.X., X.F.J., J.H.C., W.H.C., Q.L.W., and Y.H.X.: provision of study material or patients; Y.F.P., B.W., and X.Z.Y.: dataanalysis and interpretation and manuscript writing; Y.L., J.J.D., J.Y.J., X.H.Y., S.L.X., X.F.J., and W.H.S.: data analysis andinterpretation; J.M.W. and X.Z.: data interpretation and manuscript revision; X.W.B.: conception and design, financial support,administrative support, data analysis and interpretation, manuscript writing, and final approval of manuscript.

Correspondence: Xiu-Wu Bian, M.D., Ph.D., Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third MilitaryMedical University, Chongqing 400038, China. Telephone: 86-23-6875-4431; Fax: 86-23-6539-7004; e-mail: bianxiuwu@263.net Received June 1, 2011; accepted for publication November 11, 2011; first published online in STEM CELLS EXPRESS November 30,2011. VC AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1685

STEM CELLS 2012;30:108–120 www.StemCells.com

recurrence of malignant glioma. However, the state of GJICand related protein expression in GSCs remain unknown, andGJIC’s contribution to such capabilities as invasion andtumorigenicity needs to be revealed.

In this study, we first evaluated the structural basis andfunction of GJIC in GSCs isolated from either human glio-blastoma multiforme (GBM) cell line or primary gliomaspecimens and found the reduced expression of Cx proteins,Cx43 in particular, due to hypermethylated promoter regionin gap junction protein a1 (GJA1) gene (which encodesCx43). Reconstitution of Cx43 expression resulted in the inhi-bition of self-renewal, invasiveness, and tumorigenicity ofGSCs. We found that influencing the expression of E-Cad-herin and its coding proteins might be one of the underminedmechanisms. This study indicates the requirement of the lowexpression of Cx43 for maintaining GSCs in their malignantphenotype and a potential strategy for treatment of malignantglioma by upregulation of Cx43.

MATERIALS AND METHODS

Cell Line, Primary Tumors, Tumorspheres,and Adherent GSCs

Human GBM cell line U87 was purchased from American TypeCulture Collection (ATCC). Specimens from six cases of primaryhuman malignant glioma (Supporting Information Fig. S1) wereobtained clinically with the informed consent of the glioma patientsas approved by the Institutional Research Ethics Board at South-west Hospital, Chongqing, China. Tumorspheres from U87 cellsand primary specimens were isolated and characterized as previ-ously described [32, 33]. In addition, GSCs were cultured in the ad-herent condition using the method described by Pollard et al. [40].

Electron Microscopy

For observation under scanning electron microscope (SEM), thecells on coverslips after culture were fixed with 2.5% glutaralde-hyde and postfixed in 1% osmium tetroxide. After washing withphosphate-buffered saline (PBS), the samples were dehydrated bygradient ethanol. The samples were then transferred to a criticalpoint dryer in 100% ethanol and dried in CO2. Coverslips weremounted on aluminum sample stubs, gold coated by sputtering,and then observed under SEM (KYKY-EM3200 Digital SEM,Beijing, China). For examination by transmission electron micro-scope (TEM), the tumor cells were fixed with 2.5% glutaralde-hyde and postfixed in 1% osmium tetroxide. After dehydrationwith gradient ethanol and soaking in acetone, the samples werethen embedded in Epon812 resin and acetone followed by 100%Epon812 resin for 1 hour. Finally, Epon812 resin was solidified.Ultrathin sections were made with an LKB ultramicrotome(NOVA, Sweden) and stained with uranyl acetate and lead citratefor examination by TEM (JEM-2000Ex, JEOL, Japan).

Fluorescence Recovery After Photobleaching

Different groups of tumor cells were incubated with 5-carboxy-fluorescein diacetate (5(6)-CFDA, 10 lg/ml, Molecular Probes).Fine-shaped cells neighboring on three to four other cells wereselected under a laser confocal microscopy (LCM, Leica SP-5,Germany) and photobleached to 50%–90% of their original fluo-rescence intensity. The cells were examined for recovery of fluo-rescence 4 minutes later. After the transduction of Cx43, therecovery of GJIC was evaluated as follows: tumorspheres wereplated onto glass coverslips. Alexa Fluor 568 hydrazide (500 lg/ml, Molecular Probes) was loaded into tumorspheres using InfluxPinocytic Cell-Loading Reagent (Molecular Probes). After recov-ered for 10 minutes, the samples were placed under a LCM forfluorescence recovery after photobleaching (FRAP) analysis.

Immunostaining

The cells/human glioma specimens were fixed with 4% parafor-maldehyde or cold acetone and then incubated with antibodiesagainst CD133 (mouse monoclonal IgG1, Abcam, U.K.), nestin(mouse monoclonal IgG1, Chemicon), CD44s (mouse monoclonalIgG1, Neo Markers), b-tubulin III (mouse monoclonal IgG1,Chemicon), glial fibrillary acidic protein (GFAP, rabbit polyclo-nal, Dako, Denmark), myelin basic protein (MBP, rabbit polyclo-nal, Chemicon), E-cadherin (rabbit polyclonal, Santa Cruz), sexdetermining region Y-box 2 (Sox2, rabbit polyclonal, Novus Bio-logicals), Oligo2 (rabbit polyclonal, Millipore), B-cell specificmoloney murine leukemia virus insertion site 1 (Bmi-1, rabbitpolyclonal, Santa Cruz), and Cx43 (rabbit polyclonal, Zymed).Appropriate secondary antibodies (fluorescein isothiocyanate(FITC) goat anti-rabbit, tetramethyl rhodamin isothiocyanate(TRITC) goat anti-rabbit; Cy3 red goat anti-mouse; Cy5 goatanti-mouse; Molecular Probes) were used. The cells were coun-terstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma) forcell nuclei and then examined under a LCM. Immunohistochem-istry staining of tumor-bearing tissues from nude mice for nestin(Chemicon), Cx43 (mouse monoclonal IgG1, Abcam), and Ki-67(rabbit polyclonal, Zhongshan, Beijing, China) was performed bySuper Vision immunohistochemistry kit (Boster, Wuhan, China).Double-labeling immunostaining of human glioma specimens forstem cell/progenitor markers (octamer-binding transcription factor4, Oct4, rabbit polyclonal, Santa Cruz; Oligo2; Sox2) and Cx43(Abcam) was performed by DouSPTM Detection System (MaixinBiotech, Fuzhou, China) according to manufacturer’s instructions.

Reverse-Transcription Polymerase Chain Reaction(RT-PCR) and Real-Time RT-PCR

Total RNA was isolated from different cell groups by Tripure(Roche Co., Swiss), following the instructions of manufacturer.RT-PCR was carried out using RNA PCR kit 3.0 (TaKaRa, Ja-pan). The sequence of primers, the product size, and the anneal-ing temperature were shown in Supporting Information Table S1(Nos. 1–6). The results of RT-PCR were further verified by real-time RT-PCR using SYBR PrimeScript PCR kit II (TaKaRa, Ja-pan). The sequence of primers, product size, and annealing tem-perature were also shown in Supporting Information Table S1(Nos. 7–16).

Western Blotting

The equivalent amounts of protein were subject to Western blot-ting analyses. Primary antibodies, rabbit polyclonal anti-GAPDH(Beyotime, Haimen, China), Cx43 (Abcam), and E-cadherin(Santa Cruz) were used according to the manufacturer’s instruc-tions. After application of horseradish peroxidase-labeled second-ary antibodies, chemiluminescence (SuperSignal West Pico,Pierce) was quantified using ImageQuant 5.0.

Coimmunoprecipitation

Cx43 or E-cadherin pulldown assays were performed with anti-Cx43 antibody (Abcam or Santa Cruz) or anti-E-cadherin anti-body (Santa Cruz or BD, mouse monoclonal IgG1) using theDynabeads Coimmunoprecipitation Kit (Invitrogen) following themanual instructions.

Flow Cytometry

Cell suspension was pelleted and stained with anti-Cx43 for 1hour at 4�C, and then appropriate secondary antibodies (FITCGreen goat anti-rabbit; Molecular Probes) were added for 1 hourat 4�C. Analysis of fluorescence intensity was performed by flowcytometry on Coulter Epics XL (Beckman). Flow cytometry wasalso used to estimate the transduction efficacy of adenovirus vec-tor. We isolated CD133-positive cells, which were termed GSCs,from human primary glioma specimens using fluorescence-acti-vated cell sorting (FACS). Briefly, cell suspension was pelletedand stained with anti–CD133–phycoerythrin (Miltenyi Biotec,

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Germany) for 0.5 hour at 4�C, then sorted CD133-positive byflow cytometry on the FACSAria II (BD).

Promoter Methylation

Chromatin in approximately 1 � 106 cells were isolated and soni-cated by a sonicator (Diagenode, Belgium). To perform methyl-ated DNA immunoprecipitation (MeDIP), sonicated chromatinwas incubated with goat anti-human 5-methlcytidine antibodyovernight and rabbit anti-goat IgG-coupled magnetic beads for 2hours at 4�C. After elution, methyl DNA was purified. Two pairsof primers (Supporting Information Table S1, Nos. 17 and 18)were designed to amplify two potential methyl regions in GJA1gene (NM_000165) promoter. Enrichment of methylated DNAfragments obtained from the MeDIP assay was determinedby real-time PCR using SYBR PrimeScript PCR kit (TaKaRa,Japan). GAPDH (primers, Supporting Information Table S1,No. 19) served as an internal control.

Overexpression and Knocking Down

Total RNA was isolated from U87 cell line and full length ofGJA1 cDNA (AF151980) was amplified. Sequence of PCR pri-mers, products size, and annealing temperature were shown inSupporting Information Table S1 (No. 20). Then, the humanGJA1 cDNA was cloned into HindIII/BamHI sites of pAdTrack-CMV to obtain the pAdTrack-CMV-Cx43, and the prospectiverecombinant adenovirus (Ad-Cx43) was produced. Control virusAdEasy-green fluorescent protein (GFP; Ad-GFP, Mock) wasidentical to Ad-Cx43 except that it did not contain Cx43 expres-sion cassette. Viruses were amplified in HEK-293 cells. After thedetermination of virus concentration by an adenovirus titer immu-noassay kit (QuickTiter, Cell Biolabs), the suspension containingAd-Cx43 and Mock (5.0 ll/ml stem cell culture medium, 5–20multiplicity of infection) were used to infect tumor cells for atleast 72 hours. After the confirmation of transduction efficacy byflow cytometry and real-time RT-PCR, cells were collected forfurther investigation. The expression of Cx43 and E-cadherin indifferent groups was knocked down by shRNA Lentiviral par-ticles (SHGLY-NM_000165, Sigma) or siRNA (CDH1-2558,2198, 1246, Ibsbio, Co., Ltd., Shanghai, China). Sequence ofthese shRNAs and siRNAs were shown in Supporting InformationTable S2.

Proliferation Assay

Cells dissociated from different groups were prepared into singlecell suspension and seeded in 96-well plates at approximately1,000 cells per well in 0.1 ml of Dulbecco’s modified Eagle’smedium (DMEM) containing 10% FBS. On days 0, 1, 2, 3, 4, 5,6, 7, and 8, culture medium was replaced with 0.1 ml of freshDMEM containing 10% FBS and 0.01 ml Cell Counting Kit-8(CCK-8, Dojindo, Japan) solution was added. After incubation at37�C for 1.5 hours, the plates were agitated for 15 minutes, andthe optical density of the solution was measured at 490 nm in aphotometer (lQuant, Bio-TEK).

Matrigel Invasion Assay

Cells dissociated from different groups were cultured in the se-rum-free stem cell medium. On the day of experiment, the 24-well micropore polycarbonate membrane filter with 12.0 lmpores (Becton Dickinson) was coated with 10–12 ll Matrigel(Sigma, approximately 8–12 mg/ml). Then 200-ll cell suspen-sions (2 � 105 per ml) were seeded onto the top chamber in se-rum-free DMEM/F12 culture medium. The bottom chamber wasfilled with DMEM/F12 containing 10% FBS as chemoattractants.After 24 hours of incubation in a 5% CO2 humidified incubatorat 37�C, the membranes were fixed and stained by crystal violet,and the cells on the upper surface were carefully removed with acotton swab. The invasive cells on the lower surface wereobserved and counted under �200 of microscopy.

Xenografts in Nude Mice

Nude mice were purchased from the Center of Animals (ThirdMilitary Medical University, Chongqing, China). The cells disso-ciated from different groups were cultured in the serum-free stemcell medium and then injected subcutaneously (5 � 104 cells)and orthotopically (5 � 103 cells) into nude mice. Tumor-bearingtissues were fixed and cut with a vibratome horizontally into 8-lm coral sections. The sections were mounted on slides andstained with immunohistochemistry, Harris hematoxylin and alco-holic eosin (H&E). Mice were hosted according to the guidelinesof the Third Military Medical University Animal Committee.

Gene Expression Array and Gene SetEnrichment Analysis

RNA was isolated from different groups of U87 tumorsphere(Mock vs. Ad-Cx43) and labeled following manufacturer’s instruc-tions. Universal Reference RNA (Stratagene, LA Jolla, CA) waslabeled with Cy3-dUTP, and experimental samples were labeledwith Cy5-dUTP. Each labeled sample was hybridized to an Agilentwhole genome gene expression array following manufacturer’sinstruction, and the hybridized arrays were then scanned using aGenePix 4000B scanner (Molecular Devices, Sunnyvale, CA). Toanalyze sets of related genes that might be systematically altered inthe transduction of Cx43, we used a gene set enrichment analysis(GSEA), a method which includes searching for consistent butsubtle changes in gene expression by incorporating pathway anno-tations [41]. Details on the methodology for this analysis and thesoftware as well as the information on the biological data sets areavailable on the websites of http://www.broad.mit.edu/gsea andhttp://www.stanford.edu/�rnusse/pathways/targets.html. Briefly,the genes from experimental microarray were first ranked accordingto their expression differences (signal to noise ratio) betweenAd-Cx43 transduced and Mock groups. The standard GSEA nullhypothesis is that the rank ordering of the genes in a given compari-son is random and not associated with the order of the genes and/ortreatment. The extent of association was then measured by a non-parametric running sum statistic termed the enrichment score (ES),and the maximum ES (MES) was recorded over each gene set.Totally, 83 target genes of Wnt/b-catenin signaling pathway wereobserved and the enrichment profile was drawn according to the ESscore and positions of gene set members on the rank ordered list.

Statistical Analysis

Results were presented in the form of mean (X) 6 SD. The datafrom FRAP and real-time PCR were analyzed by ANOVA andStudent’s t-test. The data from primary tumorsphere formationand Matrigel invasion assay were analyzed by ANOVA. TheSPSS 10.0 statistical software was used.

RESULTS

Loss of Gap Junction-Like Structure andDysfunction of GJIC in GSCs

Tumorspheres, CD133-positive glioma cells, and GSCsexpanded in adherent culture were isolated and identifiedfrom U87 and human glioma specimens as previouslydescribed [32, 33, 40] (Supporting Information Figs. S2, S3).

The cellular surface in U87 tumorsphere was smooth withrare prominences. All cells were connected to one anothertightly. Most cells in U87 tumorspheres were in an immatureor primitive state as shown by rare organelles such as mito-chondria and endocytoplasmic reticulum. Few gap junction-like structures were found between these cells. However, gapjunction-like structures were detected among descendants dif-ferentiated from U87 tumorspheres, which contained abundantorganelles (Fig. 1A).

110 Connexin 43 Inhibits Glioma Stem Cell Invasion

FRAP is a noninvasive approach for measuring cell gapjunction coupling. In a tumorsphere, adherent non-sphere-forming cells, adherent GSCs, and descendants differentiatedfrom tumorspheres were loaded with the fluorescent dye 5(6)-CFDA, and one pulse of intense argon laser bleached the dyein a cell of interest. Subsequently, the unbleached 5(6)-CFDAin adjacent cells diffused back into the cell via gap junctions(Fig. 1B). The recovery rate in spheres (U87 tumorsphere,5.02 6 1.33%; glioma specimen-2 tumorsphere, 5.58 64.98%; glioma specimen-3 tumorsphere, 4.05 6 4.77%) wassignificantly lower than that of the controls (U87 adherentnon-sphere-forming cells, 28.32 6 7.07%, descendents differ-entiated from U87 tumorsphere, 31.43 6 7.40%; glioma spec-imen-2 adherent non-sphere-forming cells, 30.96 6 17.71%;glioma specimen-3 adherent non-sphere-forming cells, 20.146 3.87%) (Fig. 1C). The recovery rate in glioma specimen-1-derived tumorsphere was also significantly lower (6.35%).

The size of cells and the condition of cell–cell contact, inother words cell density around targeting cells, varied differ-ently between sphere and adherent cells. Thus, we examinedthe functional status of gap junction between those GSCsexpanded in adherent condition. As shown in Figure 1B and1C, the recovery rate in adherent GSCs was also low (4.00 62.79%). These results suggest that the significant reductionsin gap junction coupling in most GSCs occur not only inspheres but also in GSCs cultured under adherent conditions.

Expression of Connexins Were Lower in GSCs

We then examined the levels of Cxs, which are major compo-nents of gap junction. As shown in Figure 2, except for GJB6(Coding for Cx30, NM_006783) and GJB1 (Coding for Cx32,BC039198), the relative mRNA levels of those gap junctionprotein genes (GJB2-Cx26, NM_004004; GJA3-Cx46,NM_021954; GJA1-Cx43, AF151980; and GJA8-Cx50,NM_005267) were lower in U87 tumorspheres than in U87adherent non-sphere-forming cells (Fig. 2A, 2B), with GJA1mRNA at the lowest level. Cx43 protein level was high in ad-herent non-sphere-forming or CD133� glioma specimen cells,with considerably lower level in U87 tumorspheres orCD133þ glioma specimen cells (Fig. 2C). Flow cytometryanalysis confirmed the low expression of Cx43 in U87 tumor-spheres (Fig. 2D). Immunofluorescent staining revealed lowexpression of Cx43 in tumorspheres and adherent GSCs(derived from U87 and glioma specimens 1–3), while all cor-responding adherent non-sphere-forming cells with detectableCx43, displayed as lamellar/spot at the borders between adja-cent cells (Fig. 2E).

Hypermethylation of GJA1 Gene Promoter ReducedExpression of Cx43 in GSCs

Immunostaining for Cx43 and markers for stem cell and pro-genitors such as Oligo2, Sox2, and Oct4 in human glioma

Figure 1. Dysfunctional gap junction between GSCs. (A): Gap junction deficiency in GSCs. Although the cells in U87 tumorsphere weretightly connected (photograph of scanning electron microscopy, scale bar ¼ 10 lm), no gap junction between these cells was observed undertransmission electron microscope (TEM, scale bar ¼ 100 nm). Gap junction-like structures (denoted by red arrow) were detected under TEMbetween descendant cells differentiated from U87 tumorsphere (scale bar ¼ 200 nm). Conjunction region was denoted by yellow box. (B): Fluo-rescence recovery after photobleaching (FRAP) analysis of GJIC before reconstitution of connexins. One cell of interest (denoted by red arrow)was exposed to a short pulse laser that caused bleaching of the fluorescence. Recovered fluorescence, resulting from diffusion of unbleached dyefrom adjacent cells, was measured and plotted as a function of time when recovery was achieved. The fluorescence values were normalizedagainst a reference cell (denoted by white arrow). Scale bar ¼ 50 lm. (C): Recovery rates of different cell groups. Data from FRAP were ana-lyzed by ANOVA and Student’s t tests. Abbreviations: ANOVA, analysis of variance; GSCs, glioma stem cells.

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specimens showed that Cx43 was detected in most differenti-ated tumor cells with no expression of stem cell markers (Fig.3A, 3B); in contrast, Cx43 was not detected in the stem cellmarker-positive tumor cells. The results were consistent withthe in vitro findings.

For understanding the possible mechanism of the reducedexpression of Cx43, we examined methylation status in thepromoter of GJA1 gene. With methyl-DNA immunoprecipita-tion and real-time PCR (Fig. 3C–3E), we found that methyla-tion levels of two regions in GJA1 promoter were muchhigher in U87 tumorspheres (Fig. 3D, 3E, region 1, 6.84 �10�2; region 2, 1.03 � 10�1) than in adherent non-sphere-forming cells and differentiated descendant cells (region 1,1.96 � 10�3 and 4.49 � 10�3; region 2, 1.3 � 10�3 and 1.38� 10�4, respectively).

Reconstitution of Cx43 Inhibited In Vitro Self-Renewal,Proliferation, and Invasiveness of GSCs

An adenovirus expression vector containing GJA1 gene wasconstructed and transduced into different cell groups (Sup-porting Information Fig. S4A, S4B) with an efficacy of up to

70% (Supporting Information Fig. S4C). Enhanced expressionof GJA1 in U87 tumorspheres at mRNA and protein levelswas detected by real-time RT-PCR (Supporting InformationFig. S4D) and immunostaining (Supporting Information Fig.S4E). After the transduction of shRNA Lentiviral particles,reduced expression of GJA1 in adherent non-sphere-formingU87 cells at mRNA level was detected by real-time RT-PCR(Supporting Information Fig. S4F). Alexa Fluor 568 hydra-zide, a red fluorescent dye, was applied to evaluate the func-tional status of GJIC after the reconstitution of Cx43 (Sup-porting Information Fig. S5A–S5C). There was no significantdifference of the recovery rate between Ad-Cx43-reconstituted(Ad-Cx43) U87 tumorspheres and the control groups (Sup-porting Information Fig. S5C). U87 cells reconstituted withCx43 formed fewer primary tumorspheres in serum-free stemcell medium 7 days postseeding (1.30 6 1.42 tumorspheres/low-power field), when compared with a large quantity oftumorspheres formed by Wt U87 (16.10 6 3.57) and Mock-transduced cells (17.10 6 4.82) (Fig. 4A, 4B). The mRNAlevel of GFAP, an astrocytic differentiation marker of glioma,was increased in Ad-Cx43-transduced U87 cell line (Support-ing Information Fig. S6A). Conversely, the expression of

Figure 2. Low expression of connexins (Cxs) in GSCs. (A): Expression of Cx-coding genes detected by RT-PCR. (B): Expression of Cx-cod-ing genes detected by real-time PCR. Data were analyzed by Student’s t-test. (C): Expression of Cx43 protein detected by Western blotting.(D): Expression of Cx43 detected by flow cytometry. U87 tumorsphere group was represented by yellow curve, and negative control was repre-sented by blue curve. (E): Immunofluorescence staining of Cx43 (green/red) with the nuclei counterstained by 4,6-diamidino-2-phenylindole(blue). Scale bar ¼ 50 lm. Abbreviations: FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GJA, gap junc-tion protein a; GJB, gap junction protein b; GSCs, glioma stem cells; RT-PCR, reverse-transcription polymerase chain reaction.

112 Connexin 43 Inhibits Glioma Stem Cell Invasion

CD133, a stem cell marker, was decreased in U87 sphere af-ter the Ad-Cx43 transduction (Supporting Information Fig.S6B). Therefore, reconstitution of Cx43 in GSCs promotedcell differentiation.

The potential to proliferate by differentiation is anotherimportant property of GSCs. In DMEM þ 10% FBS, the pro-liferation of Mock U87 tumorsphere increased by 27.16-foldon day 7. However, Ad-Cx43 U87 tumorsphere showed55.50% reduction in proliferation when compared with MockU87 cells, demonstrating that Cx43 reconstitution inhibitedthe proliferation of differentiating precursor cells (Fig. 4C,4D). The histological features of the growth state of differentsphere groups on day 10 were shown in Figure 4C. Mockcells grew in multiple layers with round shape in anextremely compact appearance. However, Ad-Cx43 cells grewin a loose monolayer with elongated shapes.

Spontaneous invasion of tumor cells was reduced afterreconstitution of Cx43 (10% FBS: Wt U87 tumorsphere,199.40 6 33.92 cells/Hf; Mock-Ad U87 tumorsphere, 191.006 46.52 cells/Hf; Ad-Cx43 U87 tumorsphere, 45.80 6 12.91cells/Hf) (Fig. 4E). On the contrary, invasion of those adher-ent non-sphere-forming cells was enhanced after the knockingdown of Cx43 (10% FBS: Wt adherent non-sphere-formingU87 cells, 29.40 6 11.35 cells/Hf; Mock-Lentivirus adherentnon-sphere-forming U87 cells, 36.80 6 16.82 cells/Hf; Lenti-virus-shRNA-Cx43 adherent non-sphere-forming U87 cells,95.10 6 19.18 cells/Hf) (Fig. 4F).

Reconstitution of Cx43 InhibitedTumorigenicity of GSCs

The subcutaneous and orthotopical tumorigenicity of GSCsisolated from U87 cell line and human glioma specimens 4–6were evaluated after reconstitution of Cx43. After 8 week oftumor cell implantation, visible tumors were found in miceimplanted with Wt and Mock U87 tumorspheres. However,the size of tumors in mice receiving Ad-Cx43 U87 tumor-spheres was much smaller (Fig. 5A, 5B; Supporting Informa-tion Table S3). In the H&E-stained sections, the tissue fromAd-Cx43 group showed better differentiated tumor cells withthinner shape, less dense nuclei, and more abundant cyto-plasm than those from mock and Wt groups (Fig. 5C). Weperformed immunostaining and found less proliferation, betterdifferentiation, and increased expression of Cx43 in Ad-Cx43group compared with the controls (Fig. 5D). The orthotopicaltumorigenicity of GSCs was also inhibited after reconstitutionof Cx43. Neural symptoms and dyscrasia were observed inmice receiving Mock U87 and glioma specime 4–6 tumor-spheres, and xenografts were formed in the brains 4–8 weekspostimplantation. Under light microscopy, tumor tissues wereobserved in the brain of each mouse in this group (Fig. 5E,Supporting Information Table S3). In contrast, tumor wasdifficultly found in mouse brains implanted with 5 � 103

Ad-Cx43 transduced tumorspheres (U87 tumorspheres,glioma specimen 4–6 tumorspheres). There were significant

Figure 3. Double staining of stem cell markers/Cx43 in human primary glioma specimens and measurement of hypermethylation of GJA1 pro-moter in glioma stem cells (GSCs). (A): Fluorescent immunostaining for Cx43 (red, yellow arrows), stem cell markers Oct4, Oligo2, and Sox2(green, white arrows), with the nuclei counterstained by DAPI (blue). Scale bar ¼ 50 lm. (B): Double immunohistochemistry for Cx43 (red) andstem cell markers Oct4, Oligo2, and Sox2 (dark brown), under light microscopy �200. (C–E): Methylated DNA by immunoprecipitation foranalysis of promoter methylation. (C): Amplification regions and primers of real-time PCR. (D): Real-time PCR products from region 1. (E):Real-time PCR products from region 2. Data were analyzed by Student’s t-test. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GAPDH,glyceraldehyde-3-phosphate dehydrogenase; GJA1, gap junction protein a1; PCR, polymerase chain reaction.

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differences in survival time of the mice between the groups(Fig. 5F). These results indicated that reconstitution of Cx43profoundly inhibited the tumorigenicity of GSCs.

Reconstitution of Cx43 Upregulated E-CadherinProtein Expression

E-Cadherin is an important factor involved in the activationof Wnt/catenin signaling pathway and epithelial–mesenchymaltransition (EMT) or mesenchymal–epithelial transition, whichis crucial for invasion and stemness of cancer cells. Therefore,we examined the change of E-cadherin expression after recon-stitution of Cx43 in GSCs (spheres and adherent GSCs) byWestern blotting and immunostaining. As shown in Figure 6,E-Cadherin expression was upregulated in Cx43-reconstitutedgroups compared with the almost absence of this protein inWt and Mock groups (Fig. 6A–6C). The spatial distributionof these two proteins was observed in the adherent GSCs. Asshown in Figure 6B, we could see the localization of Cx43and E-cadherin overlapped in the cell membrane and cyto-plasm. Furthermore, the colocalization of Cx43 and E-cad-herin in human glioma specimens was also revealed by fluo-

rescent immunostaining (Fig. 6D, Supporting InformationFig. S7).

Interaction of Cx43 with E-Cadherin Resultedin the Changes of GSCs Invasiveness ViaWnt/b-Catenin Pathway

Downregulation of E-cadherin by siRNA (Supporting Informa-tion Fig. S8) could not alter the invasion of those GSCs withoutCx43 restoration (202.17 6 42.91 cells/Hf vs. 236.00 6 48.63cells/Hf) (Fig. 7A). However, for those Cx43-reconstitutingGSCs with significantly reduced invasiveness (26.00 6 12.66cells/Hf), knocking down the expression of E-cadherin withsiRNA can partially restore their invasion (101.83 6 19.43cells/Hf). These results suggest that GSC invasiveness isaffected by a possible correlation between E-cadherin and Cx43.

To gain insight into the mechanisms by which Cx43-regu-lated E-cadherin protein inhibit invasiveness/tumorigenicity ofGSCs, we performed real-time PCR and Cx43/E-cadherinpulldown assays to examine an interaction between these twogenes/proteins. We found that reconstitution of Cx43 in GSCs

Figure 4. The in vitro self-renewal, proliferation, and invasion of glioma stem cells (GSCs)/non-GSCs. (A): Formation of U87 primary tumor-sphere after the reconstitution of Cx43. (B): The number of primary tumorspheres derived from Ad-Cx43 transduced U87 cell line cells wascounted 7 days after seeding (ANOVA test). Light microscopy �100. (C): Morphology of U87 tumorsphere cells after the reconstitution of Cx43in Dulbecco’s modified Eagle’s medium þ 10% FBS for 10 days; light microscopy �200. (D): Growth curve of GSCs. (E, F): Invasion assayusing 10% FBS as stimuli. (E): Invasion of GSCs after the reconstitution of Cx43. (F): Invasion of non-GSCs after the knocking down of Cx43.Statistics analysis was done by ANOVA test. Abbreviations: ANOVA, analysis of variance; FBS, fetal bovine serum; OD, optical density;shRNA, short-hairpin RNA; Wt, wild type.

114 Connexin 43 Inhibits Glioma Stem Cell Invasion

not only changed the mRNA levels of E-cadherin (Fig. 7B)but also formed a protein complex of Cx43 with E-cadherin.As demonstrated by coimmunoprecipitation analysis(Fig. 7C), E-cadherin was detected in Cx43 immunoprecipi-tates and vice versa, Cx43 detected in E-cadherin immunopre-cipitates, whereas no signals were detected in immunoprecipi-tates of 5% inputs group.

Because E-cadherin can recruit b-catenin to the cell mem-brane by forming a complex with this protein and inhibit theactivation of Wnt/b-catenin signaling pathway [42, 43], weperformed microarray analysis to compare the expressionprofile of Wnt/b-catenin target genes between Mock andAd-Cx43 transduction groups. GSEA-P software package [41]and a list of target genes of Wnt/b-catenin signaling(http://www.stanford.edu/�rnusse/pathways/targets.html) wereapplied for interpreting gene expression data following theinstruction from inventors. Genes were ranked according totheir correlation to Ad-Cx43 transduction, and then the posi-tion of each gene-set member was identified, and a MES foreach gene set was calculated. Gene set enrichment profile andtheir corresponding heat maps are shown in Figure 7D, 7E.

Wnt/b-catenin downstream target genes were significantlylower after Ad-Cx43 transduction such as Wnt3a, Atoh1,POSIN, etc. Of note, stemness-related genes such as Sox2,Nanog, Oct4, and Sox2, which are known Wnt/b-catenindownstream target genes, were among the genes that werereduced after Ad-Cx43 transduction. Thus, it is likely thatCx43 can influence the expression of E-cadherin and form acomplex with E-cadherin to inhibit the expression of Wnt/b-catenin targeting genes, then to decrease the invasiveness/tumorigenicity of GSCs.

DISCUSSION

Cancer stem cell theory is challenging the knowledge of can-cer development and therapies. Cancer cells have been recog-nized as a heterogeneous population, even though they sharesuch malignant features as uncontrollable proliferation, poordifferentiation, and potential invasiveness. However, theresource of heterogeneity during cancer progression has notbeen clarified. Recent studies have revealed that CSCs give

Figure 5. Xenografts of glioma stem cells (GSCs) after Cx43 reconstitution. (A–C): Subcutaneous implantation of 5 � 104 U87 tumorspherecells. (A): Gross appearance of xenografts formed 9 weeks after implantation (white arrow); upper-left inset represents a tumor formed 12 weeksafter implantation. (B): The tumor sizes in different mouse groups. (C): Hematoxylin and eosin (H&E) staining, light microscopy �200. (D): Im-munostaining of subcutaneous tumor bearing tissues. (E): Orthotopical implantation of 5 � 103 GSCs in the mouse brains. H&E staining, �200under light microscopy. The tumor was indicated by blue arrow. (F): Survival curves of nude mice showing significant difference between Ad-Cx43 and Mock groups. Abbreviation: Wt, wild type.

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rise to progenitor, committed, or differentiated cancer cells,which contribute to the heterogeneity by their self-renewaland multilineage differentiation [22–24]. Therefore, CSCs arenow proposed as a crucial cell subpopulation for cancerdevelopment and malignant phenotypes including invasion,metastasis, and recurrence, and thus of importance for estab-lishment of novel treatment strategies. Malignant glioma isthe most common lethal cancer in the brain. Despite the devel-opment of neurosurgery, chemotherapy, and radiotherapy dur-ing the past decades, the mean survival time of patients withmalignant glioma has been limited within 2 years. Recent find-ings of tumor-initiating cells in glioma, that is, GSCs, provide apromising future of more effective treatment by targeting theseseeding stem cells. Therefore, investigation for potential targetof GSCs to inhibit their invasion and tumorigenicity is of signif-icant importance in translational cancer research.

In this study, we found the loss of gap junction structuresand dysfunction of GJICs in GSCs. We further found thereduced expression of gap junction proteins, especially Cx43,due to hypermethylation of its gene promoter. Most interest-ingly, we for the first time revealed that exogenous overexpres-sion of Cx43 significantly inhibited the self-renewal, tumorige-nicity, and invasiveness of GSCs, indicating that this moleculefunctions as a regulator for malignant phenotypes of GSCs. Forfurther understanding the possible mechanism, we examined theinvasion and stemness-related molecules such as E-cadherin,and found that restoration of Cx43 could upregulate and interactwith E-cadherin. Furthermore, we found that the expression ofWnt/b-catenin targeting genes was changed, which might bedue to an interaction between Cx43 and E-cadherin. The resultsmight interpret the previous findings from others that decreasedCx43 was associated with the rate of glioma cell proliferation

Figure 6. Measurement of E-cadherin protein after transduction of Cx43 in GSCs. (A): Immunostaining of U87 sphere for GFP (green), E-Cad-herin (red) under laser confocal microscope. Scale bar ¼ 10 lm. (B): Immunostaining of adherent GSCs for Cx43 (red) and E-cadherin (purple).GFP fluorescence (green) and the nuclei were counterstained by 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar ¼ 50 lm. (C): Western blot-ting for Cx43 and E-cadherin. (D): Immunostaining of glioma specimen for Cx43 (green) and E-cadherin (red). The nuclei were counterstained byDAPI (blue). Scale bar ¼ 5 lm. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GSCs, gliomastem cells; Wt, wild type.

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[14] and restoration of Cxs decreased the proliferation, tumori-genicity, and drug resistance of glioma cells [16–19].

Cx43 plays a critical role in maintaining the integrity ofgap junctions [7, 44]. Our in vitro study showed that GJIC wasdysfunctional and Cx43 was lost in CD133-positive, tumor-sphere-forming GSCs. The result was confirmed by double-immunostaining of human primary glioma specimens. In con-trast, differentiated glioma cells expressed relatively higherlevels of Cx43, forming obvious gap junctions that maintainedefficient GJIC. When GSCs were seeded into serum-containingmedium, the intracellular Cx43 level and intercellular GJICwere partially restored in the descendant cells along with theexpression of differentiation markers. Similar results wereobtained in other types of CSCs from human colon carcinomacell line HCT116 and breast cancer cell line MCF-7 (ourunpublished data) as well as CD44þ/CD24�/low breast CSCs[38, 45]. Therefore, Cx43�/low might be an additional helpfulfeature indicative of the stemness of cancer cells.

The expression and function of Cx43 are regulated by com-plicated mechanisms [46–48]. The expression of GJA1 was

regulated by promoter methylation and hypermethylation of theGJA1 promoter was detected in tumor cells [46, 47]. In thisstudy, we found hypermethylation of GJA1 promoter in GSCs,which might be one of the mechanisms to mediate suppressionof Cx43 expression in the stem cells. DNA methyltransferasessuch as Dnmt3a and Dnmt3b are directly regulated by the corepluripotency transcription factors Oct4, Sox2, and Nanog [49].This might be responsible for the phasic change of GJA1mRNA levels accompanied by the downregulation of Sox2 andOct4 during the differentiation of GSCs. This phenomenon isalso consistent with the repression of CD133, a well-knownGSC marker, caused by the promoter hypermethylation [50–52].

There is a close relationship between Cx43 and malig-nancy of gliomas [13–21]; however, the underlying mecha-nisms are still poorly understood. Furthermore, the relation-ship between the expression of Cx43 and the self-renewal,tumorigenicity, and invasiveness of CSCs remains unknown.Some signaling molecules transferred through GJIC havebeen hypothesized as the possible reason for the inhibitionof proliferation and invasion of tumor cells after the

Figure 7. Interaction of Cx43 with E-cadherin resulting in the decreased invasiveness via the Wnt/b-catenin pathway. (A): Invasion assay afterknocking down of E-cadherin (10% FBS as attractants, statistics by ANOVA test). (B): Expression of E-cadherin mRNA after Cx43 reconstitu-tion. The data were analyzed by Student’s t-test. (C): Coimmunoprecipitation. (D): Gene set enrichment analysis analysis of gene-expression pro-file for Wnt/b-catenin targets in GSCs transduced with Mock or Ad-Cx43. The top part of this plot shows the progression of the runningenrichment score and the maximum peak therein. The middle part shows the genes in Wnt/b-catenin targeting gene set as ‘‘hits’’ against theranked list of genes. The lower part shows the histogram for the ranked list of all genes in the expression data set. (E): The corresponding heatmaps show the expression values for the top subset of genes of Wnt/b-catenin targeting gene set, which contributes most to the enrichment scorein Mock group. Results are transformed into colors, where red indicates a high and blue indicates a low expression value. (F): Proposed mecha-nism of Cx43 restoration inhibiting the malignant phenotypes of GSCs. Abbreviations: ANOVA, analysis of variance; FBS, fetal bovine serum;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSC, glioma stem cells; MET, mesenchymal-epithelial transition; siRNA, small interferingRNA; TCF, T cell–specific transcription factor; IP, immunoprecipitation.

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restoration of Cx43. However, as shown in this study andothers [53, 54], the functional status of GJIC was not signifi-cantly altered between GSCs after transduction of Ad-Cx43,although the self-renewal, tumorigenicity, and invasioncapacities of these cells were obviously inhibited. Cx43 over-expression could affect EMT, a critical process involved inthe generation of CSCs and the dissemination of cancer cells[55, 56]. Cx43 via its C-terminal or cytoplasmic loop can alsoinfluence the phosphorylation of some signaling moleculesthat are critical to tumor invasion and angiogenesis [57, 58].Furthermore, Cx43 could be directed to the nucleus of cancercells given its putative nuclear targeting sequence encodedwithin the carboxyterminal domain to directly affect expres-sion of genes that are critical for self-renewal, tumorigenicity,and invasion capacities [59]. Thus, inhibition of tumor growthby Cx43 overexpression could be mediated by a mechanismthat is independent of significant GJIC.

Thus, we have investigated the underlying mechanism forCx43 to influence the malignant phenotypes of GSCs. E-Cad-herin, a glycoprotein anchoring b-catenin to cell membrane toregulate the activity of Wnt/b-catenin signaling pathway, isimplicated as a key factor in different cellular processes includ-ing invasion, stemness, and EMT [60]. Upregulation of thisprotein can inhibit the formation of b-catenin/TCF complex[43], which can initiate the transcription of a set of invasive-ness and stemness-related genes. In tumor, E-cadherin is fre-quently downregulated or extinguished and strongly enhancesthe activity of Wnt/b-catenin signaling pathway [61–65]. A se-ries of studies have revealed a possible correlation betweenCx43 and E-cadherin expression. For example, immunoelec-tron microscopic analyses showed a colocalization of Cx43 andE-cadherin at cell–cell contact sites during gap junction forma-tion [66]. As detected by immunohistochemical staining, a sig-nificant relationship between these two proteins was found invarious tumors, such as lung cancer [47] and gastric cancer[67]. Hernandez-Blazquez and Nambara et al. [68, 69] havereported that E-cadherin controls intracellular trafficking andfunction of Cx43. In addition, antibodies against E-cadherininhibited the assembly of gap junctions [70]. However, itremains unclear how these two proteins interact with eachother. In our study, reconstitution of Cx43 in GSCs could up-regulated E-cadherin expression. We further found that Cx43was clearly colocalized with E-cadherin, which is in agreementwith a previous report that extensive colocalization of Cx43and b-catenin was detected by immunocytochemistry [71]. Ofnote, knocking down the expression of E-cadherin enhancedthe invasiveness of Cx43-reconstituting GSCs.

Wnt/b-catenin pathway regulates cell fate decisions duringdevelopment and plays a central role in modulating the deli-cate balance between stemness and differentiation in severaladult stem cell niches [72, 73]. In many tissues, activation ofthis signaling has also been associated with cancer [74–76],including glioma [76]. Heterogeneous intracellular distribu-tions of those key Wnt/b-catenin signaling molecules areobserved within CSCs and their differentiated descendants. Inparticular, tumor cells located at the invasive front and thosemigrating into the adjacent stromal tissues show nuclearb-catenin staining. Hence, different levels of Wnt/b-cateninsignaling activity reflect stemness heterogeneity of tumor cell

and are likely to account for distinct cellular activities such asproliferation and invasiveness, which prompt tumor growthand malignant behavior, respectively [75].

E-Cadherin can recruit b-catenin and form a complex to in-hibit Wnt/b-catenin signaling pathway [42, 43]. We performedmicroarray analysis to compare the expression changes of Wnt/b-catenin target genes between Mock and Ad-Cx43 transduc-tion groups. Based on our results obtained from gene expres-sion array and GSEA (Fig. 7D, 7E) and some recent findingsby others [56, 71], we hypothesize that forming a complexwith E-cadherin and influencing the expression of Wnt/b-cate-nin targeting genes, such as Sox2, Nanog, Oct4, fibroblastgrowth factors (FGFs), ID2, etc., might be the candidate mech-anism for Cx43 to influence the dissemination and tumorige-nicity of GSCs (Fig. 7F). In Cx43-reduced rat epidermal kerati-nocytes, E-cadherin protein translocated from plasmamembrane to intracellular compartments. At the same time,these cells exhibited EMT features as defined by significantlymore cells expressing vimentin compared with controls [56]. Inaddition to Wnt/b-catenin pathway, both Hedgehog and Notchsignaling pathways are also important in the regulation ofstemness and invasiveness of cancer cells [77]. We found thatin the Cx43-reconstituting GSCs, the expression of both Shhand Notch2NL was downregulated by twofold but nearly two-fold expression of Notch2 was increased (data not shown).However, further analysis indicated that the activation ofHedgehog and Notch signaling pathways was hardly correlatedwith Cx43 overexpression (data not shown). Thus, Cx43 over-expression may mainly target stemness genes that are directlyregulated by Wnt/b-catenin pathway.

CONCLUSIONS

In this study, we provide evidence for the first time that dys-function of GJIC is an important feature of GSCs. Reconstitu-tion of Cx43, the key component to maintain the functionalGJIC, does not alter functional status of GJIC in GSCs butablates their self-renewal, invasiveness, and tumorigenicityalthough the interaction with E-cadherin and its coding pro-tein to downregulate the express of those Wnt/b-catenin tar-geting genes. Our results identify a potential role of Cx43 inmaintaining the malignant phenotype of GSCs.

ACKNOWLEDGMENTS

This study was supported by grants from National BasicResearch Program of China (973 Program, No. 2010CB529403)and the National Natural Science Foundation of China (NSFC,Nos. 30725035 and 30700863).

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential conflict of interest.

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120 Connexin 43 Inhibits Glioma Stem Cell Invasion